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CDKN2B Deletion Is Essential for Pancreatic Cancer Development Instead of Unmeaningful Co-Deletion Due to Juxtaposition to CDKN2A

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CDKN2B Deletion Is Essential for Pancreatic Cancer Development Instead of Unmeaningful Co-Deletion Due to Juxtaposition to CDKN2A OPEN Oncogene (2018) 37, 128–138 www.nature.com/onc ORIGINAL ARTICLE CDKN2B deletion is essential for pancreatic cancer development instead of unmeaningful co-deletion due to juxtaposition to CDKN2A QTu1,2,6, J Hao3,6, X Zhou1,2,6,LYan1,4, H Dai1, B Sun1,2, D Yang1,2,SAn1,2,LLv4, B Jiao3, C Chen1, R Lai1, P Shi3 and X Zhao1,4,5 Pancreatic cancer is among the deadliest malignancies; however, the genetic events that lead to pancreatic carcinogenesis in adults remain unclear. In vivo models in which these genetic alterations occur in adult animals may more accurately reflect the features of human cancer. In this study, we demonstrate that inactivation of Cdkn2b (p15ink4b) is necessary for induction of pancreatic cancer by oncogenic KRASG12D expression and inactivation of Tp53 and Cdkn2a in adult mouse pancreatic ductal cells (P60 or older). KRASG12D overexpression in these cells activated transforming growth factor-β signaling and expression of CDKN2B, which, along with CDKN2A, led to cellular senescence and protected cells from KRAS-mediated transformation via inhibition of retinoblastoma phosphorylation. These results show a critical role of CDKN2B inactivation in pancreatic carcinogenesis, and provide a useful adult animal model by genetic engineering via lentiviral delivery. Oncogene (2018) 37, 128–138; doi:10.1038/onc.2017.316; published online 11 September 2017 INTRODUCTION pancreatic cancer result from accumulation of mutations during Only 6% of pancreatic cancer patients survive longer than 5 years adulthood, in vivo models induced by genetic engineering in adult after initial diagnosis.1,2 Kirsten rat sarcoma (KRAS), tumor protein animals may more accurately reflect the features of human cancer. (TP)53 and cyclin-dependent kinase inhibitor (CDKN)2A are the most To this end, in this study we established a pancreatic cancer 3–5 model in mouse elder than P60 by lentiviral delivery to express frequently mutated genes in human pancreatic cancer. Expres- G12D sion of oncogenic KRAS during development induces postnatal oncogenic KRAS and short hairpin (sh)RNAs targeting tumor- pancreatic intraepithelial neoplasia (PanIN), which occasionally suppressor genes frequently mutated in human pancreatic cancer. We found that pancreatic cancer was induced in adult mice by the progresses to pancreatic cancer with long latency. Inactivation of G12D Tp53, Cdkn2a and/or mothers against decapentaplegic homolog combination of KRAS overexpression and loss of Tp53 and Cdkn2a only if Cdkn2b was concomitantly inactivated. Moreover, (Smad)4 tumor-suppressor genes has been shown to promote G12D 6–8 CDKN2B expression was induced by oncogenic KRAS via cancer progression and metastasis. β Most pancreatic cancer models have been generated by transforming growth factor (TGF)- signaling. Finally, inactivation overexpressing oncogenic KRAS and inactivating Tp53, Cdkn2a, of both Cdkn2b and Cdkn2a was necessary for Rb phosphorylation and other tumor suppressors such as retinoblastoma (Rb).9–14 The and to encompass oncogene-induced cellular senescence. most widely used pancreatic cancer models are KC model developed in LSL-KrasG12D/Pdx1-Cre mice15 and KPC model developed in LSL-KrasG12D/LSL-tp53R172H;Pdx1-Cre.8 Expression of RESULTS KRAS and Tp53 mutants were initiated since early embryonic Induction of pancreatic cancer in adult mice stage in these models. Recently some models were reported to be A search of The Cancer Genome Atlas (TCGA) revealed that 10 induced in adulthood before P60 (postnatal day 60). However, it alterations in KRAS, TP53 and CDKN2A are the most frequent was reported that the capacity of KRAS to induce PanIN in mouse genetic aberrations in human pancreatic cancer (86%, 70% and pancreatic acinar cells decreases with age and is completely 52% of cases, respectively) (Supplementary Figure 1). Similar data abolished after P60.10 Moreover, KRASG12D expression in mature are reported in other cancer genome databases.17,18 We therefore acinar cells did not induce obvious lesions even in combination decided to test if pancreatic cancer can be induced by with Tp53 or Cdkn2a deficiency.10 Adult rat acinar cells also dysregulating KRAS, TP53 and CDKN2A genes via lentiviral showed resistance to KRAS-induced transformation,16 whereas infection in 9-week-old mice, which were previously reported to pancreatic ductal cells were 4100 times more resistant to be resistant to pancreatic cancer induction.10 The lentiviral vectors transformation.13 Given that most human malignancies including used in this study are illustrated in Supplementary Figure 2a. 1Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, Kunming Institute of Zoology, Kunming, China; 2Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, China; 3State Key Laboratory of Genetic Resources and Evolution, Laboratory of Evolutionary and Functional Genomics, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, China; 4Kunming Primate Research Center, Chinese Academy of Sciences, Kunming, China and 5KIZ-SU Joint Laboratory of Animal Model and Drug Development, College of Pharmaceutical Sciences, Soochow University, Suzhou, China. Correspondence: Dr X Zhao or Dr P Shi, Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences/Key Laboratory of Bioactive Peptides of Yunnan Province, Kunming Institute of Zoology, 32 East Jiaochang Road, Kunming, Yunnan 650223, China. E-mail: [email protected] or [email protected] 6These authors contributed equally to this work. Received 24 January 2017; revised 7 June 2017; accepted 31 July 2017; published online 11 September 2017 CDKN2B expression induced by KRAS inhibits pancreatic cancer QTuet al 129 Lentiviral delivery of KRASG12D along with shRNA targeting Tp53 Figure 2b). To exclude the possibility that Cdkn2a knockdown (KRAS-shTp53) was used as negative control because KRASG12D efficiency was insufficient to induce tumor formation, we injected plus p53 knockout were reported to fail to induce cancer in adult. KRAS-shTp53 lentivirus into the pancreas of adult Cdkn2a− / − mice However, lentiviral delivery of KRASG12D along with shRNAs and still failed to develop tumors (Figure 1a). targeting Tp53 and Cdkn2a (KRAS-shTp53-shCdkn2a) into adult CDKN2B encodes p15INK4B, which activates the Rb pathway pancreas failed to induce pancreatic cancer (Figure 1a), although and is co-deleted with CDKN2A in many cancers, including the Tp53-specific shRNA cassette has been used to induce tumors pancreatic cancer (Supplementary Figure 1). We therefore in previous studies19,20 and the knockdown efficiency of Cdkn2a- investigated the role of the Cdkn2b gene in pancreatic cancer. specific shRNA targeting both p16Ink4a and p19Arf sequences was We verified the knockdown efficiency of shRNA targeting Cdkn2b confirmed in mouse embryonic fibroblasts (Supplementary (Supplementary Figure 2b). KRAS-shTp53-shCdkn2b lentivirus Figure 1. Oncogenic KRASG12D expression together with shRNA-mediated inactivation of TP53, CDKN2A and CDKN2B induced pancreatic cancer. (a) Survival curve. Nine-week-old male mice with C57B6/L background were injected with lentivirus on day 0. KRAS-shTP53 group was used as control. Only the mice in the KRAS-shTP53-shCDKNB/CDKN2A − / − and KRAS-shTP53-shCDKN2A/B group developed tumors. (b) Hematoxylin and eosin (H&E) staining of paraffin sections of normal and cancerous pancreatic tissues. The arrows in the image of the normal tissue indicates a normal duct and ‘is’ denotes pancreatic islet. The arrows in cancer tissue indicate the glandular structures and the asterisk indicate fibrous stroma. Scale bars: 40 μm. (c) Mouse pancreatic cancer express established molecular markers of human pancreatic cancer. The proportion of Ki67-positive cells in pancreatic cancer tissues indicates a high proliferation index. Scale bars: 40 μm. Oncogene (2018) 128 – 138 CDKN2B expression induced by KRAS inhibits pancreatic cancer QTuet al 130 failed to induce pancreatic cancer in adult wild-type mice; Pancreatic cancers originate from pancreatic ductal cells however, simultaneous inactivation of Cdkn2a and Cdkn2b, either To identify the cell-of-origin of pancreatic cancer in our model, by injecting the KRAS-shTp53-shCdkn2b lentivirus into the enhanced green fluorescent protein (EGFP)-expressing lentivirus − − pancreas of adult Cdkn2a / mice or the KRAS-shTp53- was injected into the pancreas of adult mice. Infected (EGFP- shCdkn2a-shCdkn2b lentivirus into Cdkn2a wild-type mice, positive) cells were detected 7 days after injection. In addition to resulted in pancreatic cancer development at 4 weeks post- the lentivirus shown in Supplementary Figure 2a, we also injected injection (Figure 1a). the empty pTomo vector expressing EGFP to exclude the Pancreatic tumors induced by lentivirus KRAS-shTp53-shCdkn2b possibility that the introduced genes induced a change in cell in Cdkn2a− / − mice or by KRAS-shTp53-shCdkn2a/b in wild-type type. Although pancreatic cells are not sensitive to lentiviral 21 mice showed similar morphology, ranging in size from 0.5 to infection, we found that mostly ductal cells were infected as 1.5 cm, with yellowish white cut surfaces and few signs of demonstrated by CK-19 expression, whereas only a few acinar hemorrhage or necrosis. The tumors were frequently observed to cells were EGFP positive (Figures 2a and b and Supplementary obstruct the common bile duct and/or the main pancreatic duct,
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